Developmental Biology 294 (2006) 83 91 www.elsevier.

com/locate/ydbio

Pollen-specific pectin methylesterase involved in pollen tube growth

Guo-Wei Tian, Min-Huei Chen, Adi Zaltsman, Vitaly Citovsky
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794-5215, USA Received for publication 29 September 2005; revised 15 February 2006; accepted 16 February 2006 Available online 27 March 2006

Abstract Pollen tube elongation in the pistil is a crucial step in the sexual reproduction of plants. Because the wall of the pollen tube tip is composed of a single layer of pectin and, unlike most other plant cell walls, does not contain cellulose or callose, pectin methylesterases (PMEs) likely play a central role in the pollen tube growth and determination of pollen tube morphology. Thus, the functional studies of pollen-specific PMEs, which are still in their infancy, are important for understanding the pollen development. We identified a new Arabidopsis pollen-specific PME, AtPPME1, characterized its native expression pattern, and used reverse genetics to demonstrate its involvement in determination of the shape of the pollen tube and the rate of its elongation. 2006 Elsevier Inc. All rights reserved.
Keywords: Arabidopsis; Native PME expression; Pectin; Pollen grain; Pollen tube

Introduction Pollen tube elongation in the pistil is a crucial step in the sexual reproduction of plants. The pollen tubes invade the stigmatic tissue, penetrate the style, and eventually deposit the two sperm cells in the embryo sac where they fuse with the egg and central cell to form the zygote and endosperm, completing the fertilization process (reviewed in Lord and Russell, 2002; Palanivelu and Preuss, 2000; Preuss, 2002). While the pollen tube growth is known to occur exclusively at the tip with the new tube wall continuously forming at the growing tip (Taylor and Hepler, 1997), the molecular mechanism of this growth process remains obscure. The wall of the pollen tube tip is composed of a single layer of pectin and, unlike many other plant cell walls, does not contain cellulose or callose (Ferguson et al., 1998; Li et al., 1994). Thus, pectin metabolism and modification likely play a central role in the pollen tube growth. Pectins are polymerized in the Golgi, methylesterified and modified with side chains, and subsequently released into the apoplastic space as highly methylesterified polymers. The homogalacturonan component of pectin can later be demethylesterified by pectin methylesterases (PMEs) (Micheli, 2001). This enzymatic activity of PMEs can lead either to cell wall
Corresponding author. Fax: +1 631 632 8575. E-mail address: vitaly.citovsky@stonybrook.edu (V. Citovsky). 0012-1606/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2006.02.026

loosening or to cell wall stiffening, depending on the apoplastic pH (Catoire et al., 1998; Dens et al., 2000; Micheli, 2001). In higher plants, pectin demethylesterification is catalyzed by a number of PME isoenzymes which can express their activities in response to certain developmental or environmental cues and/ or in a tissue-specific fashion. For example, while some PMEs are ubiquitously present (Gaffe et al., 1997), others are specifically expressed during root development (Wen et al., 1999), fruit ripening (Brummell and Harpster, 2001; Frenkel et al., 1998), or stem elongation (Bordenave et al., 1996; Pilling et al., 2000). Furthermore, recent analysis of pollen-specific transcriptome of Arabidopsis indicated that several PMEs are specifically expressed in floral buds, including pollen (Pina et al., 2005). However, despite the apparently major role that PMEs may play in the growth of the pollen tube the cell wall of which is composed mainly of pectins the functional studies of pollen-specific PMEs are still in their infancy. To date, only two studies examined the PME function during pollen development and pollen tube growth; specifically, exogenously added tobacco PME has been shown to inhibit pollen tube growth by thickening the apical cell wall (Bosch et al., 2005) whereas the inactivation of VANGUARD1 (VGD1), the only Arabidopsis PME with a demonstrated function in the pollen tube growth, resulted in unstable and poorly growing pollen tubes (Jiang et al., 2005). The diversity of Arabidopsis pollen-expressed PMEs (Pina et al., 2005) suggests that

84

G.-W. Tian et al. / Developmental Biology 294 (2006) 8391

additional members of this protein family may be involved in pollen tube growth, potentially affecting different aspects of this process. Here, we identified a new Arabidopsis pollen-specific PME, AtPPME1, characterized its native expression pattern, and used reverse genetics to demonstrate its involvement in determination of the growth rate and the shape of the pollen tube.
Materials and methods Plant material
All Arabidopsis thaliana plants used in this study were in the Col-0 background. Plants were grown in soil at 22C under a light cycle of 16 h light/ 8 h dark.

Genetic complementation of the atppme1 mutant

To produce the full-length AtPPME1 transgene, we utilized a modified FTFLP technique (Li et al., 2005; Tian et al., 2004). The full-length AtPPME1 gene with its native regulatory elements was amplified using the P1/P4 primers (see above), and then the attB1 and attB2 recombination sites were added using the Gateway primers as described above. The amplified AtPPME1 gene was recombined into pDONR207, transferred to the binary destination vector pSAT6-DEST, a derivative of pSAT6-DEST-EGFP-C1 (Tzfira et al., 2005) without the GFP sequence, and then subcloned into the pRCS2-hpt binary vector (Tzfira et al., 2005). The resulting binary construct was used to produce transgenic plants as described above, except that the transformants were selected on a hygromycin-containing medium.

RT-PCR
Total RNA was extracted from 2.0 g of plant tissues, using TRI REAGENT (Molecular Research Center, Inc., Cincinnati, OH), treated with RQ1 RNasefree DNase (Promega, Madison, WI), and reverse-transcribed with Moloney murine leukemia virus (M-MLV) reverse transcriptase using the dT23VN primer (New England Biolabs, Beverly, MA). The resulting cDNAs were PCRamplified (Kang et al., 1995; Ni et al., 1998) using a mixture of AtPPME1specific primers and primers specific for the Arabidopsis ACT8 actin gene (At1g49240, An et al., 1996). AtPPME1-specific forward 5-cggcaccagatggtaaaact-3 and reverse 5-tggatgtagccgagggatag-3 primers generated a 559-bp product from the AtPPME1 transcript, while actin-specific forward 5accttgctggtcgtgacctt-3 and reverse 5-gatcccgtcatggaaacgat-3 primers generated a 632-bp product from the ACT8 transcript.

YFP tagging and native expression of AtPPME1

Transgenic Arabidopsis plants expressing YFP-tagged AtPPME1 from its native regulatory sequences were generated using the fluorescent tagging of full-length proteins (FTFLP) technique (see http://aztec.stanford.edu/gfp/ and Tian et al., 2004). We constructed a transgene containing the native AtPPME1 5 UTR with the promoter, coding region with introns, and the 3 UTR sequences; because most regulatory sequences in Arabidopsis are contained in relatively small (2 kb) regions (The Arabidopsis Genome Initiative, 2000), we included 1516 bp upstream of the AtPPME1 translation initiation codon and 1000 bp downstream of the STOP codon in our constructs. First, AtPPME1 was amplified from genomic DNA purified from leaves of 6-week-old plants using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA) as two fragments using two sets of primers, P1/P2 and P3/ P4. The sequences of these primers were P1: 5-gctcgatccacctaggctcatgtaaacaaatagcag-3, P2: 5-cacagctccacctccacctccaggccggcctccttggatgtagccgag-3, P3: 5-tgctggtgctgctgcggccgctggggcctccaagtggcttctcccacc-3, and P4: 5cgtagcgagaccacaggaagcagagagagtaacacc-3. For the second PCR reaction, a pair of genenon-specific Gateway (Walhout et al., 2000) primers were designed. The forward primer, 5-ggggacaagtttgtacaaaaaagcaggctgctcgatccacctaggct-3, contained the attB1 sequence, and the reverse primer, 5ggggaccactttgtacaagaaagctgggtcgtagcgagaccacagga-3, contained the attB2 sequence. These primers were combined with three templates, i.e., the YFP sequence derived from pRSETB-Citrine (Griesbeck et al., 2001) and two AtPPME1 fragments, and a triple template PCR (TT-PCR) was performed to produce the full-length AtPPME1 gene with the YFP coding sequence inserted into its last exon 30 bp upstream of the STOP codon (Tian et al., 2004). All PCR reactions were performed using the ExTaq DNA polymerase (TaKaRa, Japan) which, under our conditions, exhibited the highest fidelity while still capable of efficiently amplifying long DNA fragments (Tian et al., 2004). The resulting TT-PCR product was recombined into the Gateway donor vector pDONR207 (Invitrogen), verified by DNA sequencing, and transferred by Gateway recombination into the binary destination vector pBIN-GW (Tian et al., 2004). Finally, the binary construct was introduced into the Agrobacterium tumefaciens strain GV3101, and the resulting bacterial cultures were used to transform Arabidopsis plants by the modified flower dip method (Kim et al., 2003). Seven kanamycin-resistant T1 transformants were selected for analysis of the YFP-tagged AtPPME1 expression.

In vitro pollen tube growth

Fully open flowers with freshly dehiscent anthers were collected and airdried for 24 h. Pollen grains were germinated on the glass slides with agarose pads containing 10% sucrose, 0.01% boric acid, 3 mM Ca(NO3)2 (Thorsness et al., 1993), and 1% agarose in a moist kimwipe box incubated at 28C (JohnsonBrousseau and McCormick, 2004). The growing pollen tubes were examined and their length measured 6 h and 24 h after beginning of germination. For length measurements, 150 pollen tubes of each sample were chosen randomly.

Quantification of the PME enzymatic activity

PME activity was quantified by a gel diffusion assay (Bourgault and Bewley, 2002; Downie et al., 1998) as described (Chen and Citovsky, 2003) with modifications. Briefly, 100 mg of pollen grains was collected using a vacuum cleaner method (Johnson-Brousseau and McCormick, 2004) from the wild-type, mutant, and genetically complemented plants, placed in a 1.5-ml microfuge tube containing one 3-mm glass bead and 400 l of extraction buffer [1 M NaCl, 2.5 mM phenylmethylsulfonyl fluoride (PMSF), 10 M leupeptin, 0.1 M citrate/ 0.2 M sodium phosphate, dibasic, pH 7.0] and homogenized for 10 s at 4C in a TPC M250 Amalgamator (TPC Advance Technology, CA). The homogenate was cleared (20,000 g for 10 min at 4C), and the protein content of the supernatant was quantified by the Bradford method (Ausubel et al., 1987). Protein samples (50 g) were loaded into 2-mm round wells in a 2% (w/v) agarose gel containing 0.1% of 90% esterified citrus fruit pectin (Sigma-Aldrich) and 0.5 mg/ml EIA-grade gelatin (BioRad, CA) in a Petri dish (pectin served as PME substrate and gelatin was used to further stabilize the tested pollen extracts; Bourgault and Bewley, 2002; Downie et al., 1998). The gels were incubated for 16 h at 30C, rinsed with water, stained for 45 min at 25C with 0.05% (w/v) ruthenium red (Sigma-Aldrich) which stains deesterified pectin (Downie et al., 1998), and the diameter of each stained zone was measured to the nearest 0.1 mm with calipers. The PME activity in nanokatals (nkatal) was calculated based on the standard curve of log-transformed enzyme activity versus stained zone diameter generated using a commercial-grade orange peel PME (Sigma-Aldrich).

Identification of AtPPME1 homozygous lines

T-DNA insertion line SALK_077776 of Arabidopsis (Alonso et al., 2003) was received from the Arabidopsis Biological Resource Center (Ohio State University, Columbus). Plants homozygous for T-DNA insertion in the AtPPME1 gene were identified by PCR using genomic DNA extracted with the DNeasy Plant Mini Kit, two gene-specific primers, 5-atcacccgctttgtgttccct3 and 5-tgtcctcttgattgcagcttcttc-3, and a T-DNA left border-specific LBb1 primer, 5-gcgtggaccgcttgctgcaact-3 as described by the Salk Institute Genomic Analysis Laboratory (SIGNAL, http://signal.salk.edu/tdnaprimers.html).

Microscopy
For imaging of pollen in transgenic plants expressing YFP-tagged AtPPME1, mature anthers or pistils from fully open flowers were collected on

G.-W. Tian et al. / Developmental Biology 294 (2006) 8391 Table 1 Arabidopsis PMEs Table 1 (continued)

85

(a) Corresponds to the AtPPME1 gene.

glass slides and mounted in water. Plant cell walls and pollen grain walls were visualized by staining with 10 g/ml propidium iodide. For imaging of in vitro pollen tube growth, the pollen tube samples were mounted in the medium composed of glycerol/water (1:1 v/v). Confocal images were collected with a Zeiss LSM 5 Pascal laser scanning confocal microscope system. A 514 nm argon laser was used to excite YFP, and a reflected light coupled with DIC condenser was used for phase contrast digital imaging. The length of the pollen tubes was determined using the semi-automatic Profile function of the LSM Zeiss Pascal 5 software, and the average rate of pollen tube elongation determined over 24 h of growth.

Results Arabidopsis PMEs The Arabidopsis genome encodes 66 PME-related proteins (CAZY database, http://afmb.cnrs-mrs.fr/CAZY/). Amino acid sequence analysis of these proteins using the SMART tool (Letunic et al., 2004; Schultz et al., 1998) showed that 23 of them contain only the catalytic PME domain (Markovic and Jornvall, 1992; Plastow, 1988; Ray et al., 1988), while 43 also contain a PME inhibitor (PMEI) domain (Camardella et al., 2000; Giovane et al., 1995) (Table 1). Thus, Arabidopsis PMEs can be subdivided into two general groups based on their domain structure (Table 1); group 1 includes smaller proteins with 250400 amino acid residues and molecular mass of 3045 kDa that contain a PME domain, and group 2 comprises larger proteins with 500 700 amino acid residues and molecular mass of 55105 kDa that include, in addition to a PME domain, 13 PMEI domains. Because the one Arabidopsis PME, VGD1, known to function in pollen tube growth belongs to group 2 (Jiang et al., 2005), we decided to focus on the members of group 1, the role of which in pollen tube development has not been examined. Fig. 1A shows an unrooted phylogenetic tree for full-length amino acid sequences of the group 1 PMEs which revealed the presence of three clusters containing 13, 6, and 4 genes, respectively. One of these genes, At1g69940 which encodes a 361-aa protein containing a conserved PME domain and an N-terminal signal peptide (Fig. 1B) was not included in the recent Arabidopsis pollen transcriptome studies (Pina et al., 2005). Thus, we selected it for detailed expression and reverse genetics analyses. Pollen-specific expression of AtPPME1 To examine potential tissue specificity of the At1g69940 gene expression, we utilized the Fluorescent Tagging of FullLength Proteins (FTFLP) technique (Tian et al., 2004). In this approach, which has been shown to faithfully reproduce the

86

G.-W. Tian et al. / Developmental Biology 294 (2006) 8391

anther (Figs. 2A, B), indicating highly restrictive expression of this gene (Figs. 2A, B), which we therefore named AtPPME1 (A. thaliana pollen-specific PME 1). Within the expressing pollen grains, AtPPME1 accumulated mainly around the cell periphery and in intracellular vesicles, potentially representing the ER and/or Golgi endomembrane system (Fig. 2C); this subcellular localization is consistent with the presence of the signal peptide in AtPPME1 and the known secretion pathways of PMEs in plant cells (Micheli, 2001). During fertilization, pollen grains are deposited on the stigma, and the compatible interaction between pollen and stigmatic cells triggers growth of the pollen tube which invades the stigmatic tissue, ultimately reaching the ovule (reviewed in Lord and Russell, 2002; Palanivelu and Preuss, 2000; Preuss, 2002). Fig. 3 shows that the growing pollen tube exhibited high

Fig. 1. Phylogenetic analysis of group 1 Arabidopsis PMEs and amino acid sequence of AtPPME1 and its protein product. (A) Phylogenetic tree constructed by the neighbor-joining method (Saitou and Nei, 1987) using the ClustalW algorithm (http://www.genebee.msu.su/clustal/advanced.html). The gene for AtPPME1 is highlighted in orange. (B) Amino acid sequence of AtPPME1. The N-terminal signal peptide is shown in lowercase and highlighted in green and the PME domain is highlighted in orange. Numbers on the left indicate positions of amino acid residues.

native level and pattern of gene expression as well as subcellular localization of the fluorescently tagged gene product (Chen et al., 2005; Tian et al., 2004), the At1g69940 gene was amplified to include its complete intergenic regions i.e., the 5 UTR and promoter sequences and the 3 UTR and downstream sequence and the coding region with introns and with a YFP tag inserted 30 bp (10 amino acids) upstream of the stop codon (Tian et al., 2004). The location of the tag was chosen outside of the PME domain and distant from the signal peptide (see Fig. 1B). This expression construct was introduced into Arabidopsis by Agrobacterium-mediated transformation, and several lines of the resulting transgenic plants were analyzed by confocal microscopy. We observed no expression of the full-length At1g69940 gene driven by its native promoter and tagged with YFP in any of the plant organs and tissues (data not shown) except for mature pollen (Fig. 2). The YFP signal was detected only in the pollen grains, but not in the numerous surrounding cells of the

Fig. 2. Pollen-specific expression of YFP-tagged AtPPME1. All panels show mature pollen grains in anther sacs at different magnifications. YFP signal is in green, and plastid autofluorescence is in red. All images are single confocal sections. Scale bars = 30 m.

G.-W. Tian et al. / Developmental Biology 294 (2006) 8391

87

Fig. 3. Expression of YFP-tagged AtPPME1 in pollen grains and pollen tubes. (A, B) Pollen grains and pollen tubes growing between stigmatic papillae on stigma in a living pistil. Cell walls were stained with propidium iodide. (C) YFPAtPPME1 expression in a pollen tube germinated and grown for 24 h in vitro. (D) Phase contrast image of the pollen tube shown in panel C. (E) Merged images from panels C and D. YFP signal is in green, and plastid autofluorescence and propidium iodide signal are in red. Images in panels A and B are projections of several confocal sections, image in panel C is a single confocal section. Scale bars = 10 m.

levels of the AtPPME1 expression on stigma (panels A, B) and in isolated pollen grains germinated in vitro (panels CE). Consistent with the previous observations with the VGD1 PME (Jiang et al., 2005), the YFP-tagged AtPPME1 was distributed throughout the whole pollen tube (Figs. 3CE). Again, confirming its pollen-specific expression, the YFP-tagged AtPPME1 was found only in the growing pollen, but not in the adjacent stigmatic papillae (Figs. 3A, B). AtPPME1 is involved in pollen tube growth The highly specific expression pattern of AtPPME1 suggests that this protein exerts its main biological effects during pollen development and/or pollen tube growth. This notion was confirmed by our reverse genetics analyses of an AtPPME1

knockout plant line. To this end, we mined the SALK collection of Arabidopsis mutants (Alonso et al., 2003) and selected a mutant with a T-DNA insertion(s) in the second exon of the AtPPME1 gene. The mutant plants were self-pollinated, the homozygous line was identified by PCR analysis, and the location of the 5-end of the T-DNA insert in this line, designated atppme1, was confirmed by DNA sequencing (not shown). We then used RT-PCR analysis to confirm that the atppme1 plants do not contain the transcripts of the mutated gene. Fig. 4A shows that wild-type Arabidopsis plants accumulated the 559-bp RT-PCR product corresponding to the AtPPME1 transcript (lane 1, bottom band) in their floral tissues. This expression was not observed in other plant tissues, such as roots (not shown), leaves (lane 2), or stems (lane 3), consistent with the pollen-specific pattern of expression of the YFP-tagged AtPPME1 (see Figs. 2 and 3). In contrast, no RT-PCR products specific for the AtPPME1 transcript were detected in atppme1 plants (Fig. 4, lanes 46), indicating that this mutant line indeed represented a null, knockout mutant for the AtPPME1 gene. In control experiments, analysis of actin-specific transcripts generated similar amounts of RT-PCR products in all samples, indicating equal efficiencies of the RT-PCR reactions (Fig. 4A, upper bands in all lanes). The lack of the AtPPME1 expression resulted in reduction of the overall PME activity in pollen grain of the atppme1 plants. Fig. 4C shows that the ability of mutant pollen to demethylestrify pectins (PME activity = 680 nkatal/mg protein) was decreased by 20% as compared to the wild-type pollen (PME activity = 850 nkatal/mg protein). This reduction was comparable to that obtained in a knockout mutant of another pollenexpressed PME, VGD1 (Jiang et al., 2005), and it supported the notion that the AtPPME1 gene indeed encodes a functional PME enzyme. Next, we investigated the effects of the absence of AtPPME1 on pollen growth and morphology in the atppme1 plants. To this end, we germinated and cultured pollen grains in vitro. This approach allowed us to clearly characterize the morphology of the growing pollen tubes, and to precisely determine the rate of the growth. Three types of Arabidopsis plants were used in these experiments: wild-type plants, transgenic plants expressing YFP-tagged AtPPME1, and the atppme1 knockout mutant. Wild-type pollen tubes grew straight (Fig. 5A) and were indistinguishable from those of the YFP-AtPPME1 plants (not shown). In contrast, already early in the growth process, i.e., 6 h after germination, pollen tubes of the atppme1 mutant, germinated under the same conditions, exhibited curved, irregular morphology and were dramatically stunted (compare Figs. 5B D to Fig. 5A). The mutant pollen tubes remained dramatically shorter than the wild-type ones as the growth continued, i.e., 24 h after germination (compare Fig. 5F to Fig. 5E). Higher magnification of the growing pollen tube tip showed that, in the atppme1 knockout mutant, it has lost the characteristic relatively regular shape (compare Figs. 5H to I). The shorter length of pollen tubes in the atppme1 plants was reflected by the reduced rate of the pollen tube growth. Specifically, quantification of the pollen tube growth showed that the average rate of tube elongation in the atppme1 mutant

88

G.-W. Tian et al. / Developmental Biology 294 (2006) 8391

Fig. 4. RT-PCR detection of AtPPME1 transcripts and quantification of PME activity in pollen. (A) Wild-type (lanes 13) and mutant atppme1 plants (lanes 46). (B) Mutant atppme1 plants genetically complemented with the wild-type AtPPME1 gene. Lanes 1 and 4, flower; lanes 2 and 5, leaves; lanes 3 and 6, stems; lane M, molecular size markers. RT-PCR products specific for the AtPPME1 transcript (559 bp) and for the ACT8 transcript (632 bp) are indicated on the left. Molecular size markers in base pairs (bp) are indicated on the right. (C) PME enzymatic activity in pollen. Black, gray, and white bars indicate PME activity in the wild-type and atppme1 plants and in atppme1 plants genetically complemented with the wild-type AtPPME1 gene, respectively. All data represent average values of three independent measurements with indicated standard deviation values.

(10.4 3.3 m/h, n = 150) was significantly lower than that in the wild-type plants (31.8 5.0 m/h, n = 150). Interestingly, AtPPME1 specifically affected the pollen tube growth, length, and morphology, but it had no apparent effect on the morphology of ungerminated pollen grains and their cell walls pattern (Fig. 6) or on the efficiency of pollen germination in vitro. Similarly, we observed no differences between the wild-type and the homozygous atppme1 mutant plants in their seed production and fertility. Specifically, both plant lines contained similar numbers of seeds in their siliques, i.e., 37.8 2.9 (n = 50) per silique for the wild-type plants and 37.6 3.1 (n = 50) per silique for the homozygous atppme1 plants. When heterozygous atppme1 male plants were genetically crossed with wild-type female plants, the resulting F1 progeny contained an approximately 1:1 mixture (14:13, n = 27) of heterozygous to wild-type seed genotypes in their siliques. Consistently, when heterozygous atppme1 plants were selfpollinated, their F1 progeny segregated as 1:2:1 (6:13:6 atppme1 homozygous to heterozygous to wild-type ratio, n = 23). Collectively, these data suggest that, although irregular and

stunted, the atppme1 pollen tubes largely retain their functionality during fertilization. To exclude this possibility that another, unrelated mutation may have contributed to the atppme1 phenotype, we performed a genetic complementation analysis. The atppme1 mutant was transformed with a transgene corresponding to the full-length genomic sequence of AtPPME1 with its native regulatory elements. The resulting plants expressed the AtPPME1 transgene, accumulating its transcripts as detected by RT-PCR (Fig. 4B). This expression occurred in floral tissues (Fig. 4B, lane 1, lower band), but not in leaves (lane 2), stems (lane 3), or roots (not shown), mirroring expression of the wild-type AtPPME1 (see Fig. 4A). Analysis of actin-specific transcripts produced similar amounts of PCR products in all samples, indicating equal efficiencies of the RT-PCR reactions (Fig. 4B, upper bands in all lanes). We then examined the morphology and growth rate of the pollen tubes derived from the atppme1 mutant plants that expressed the AtPPME1 transgene from its native regulatory sequences. These transgenic plants developed pollen grains that, when germinated in vitro, formed pollen tubes indistinguishable

G.-W. Tian et al. / Developmental Biology 294 (2006) 8391

89

Discussion PMEs must play a central role in growth of pollen tube wall which is composed mainly of pectin (Ferguson et al., 1998; Li et al., 1994). Indeed, PME-related genes are expressed in pollen of many diverse plant species, e.g., Brassica (Albani et al., 1991), alfalfa (Qiu and Erickson, 1995), maize (Wakeley et al., 1998), tobacco (Bosch et al., 2005; Lacoux et al., 2003; Rogers et al., 2001), Salix (Futamura et al., 2000), Medicago (RodrguezLlorente et al., 2004), and Arabidopsis (Jiang et al., 2005; Pina et al., 2005). Exploration of the specific effects of PMEs on the pollen tube growth, however, is just the beginning, with one study showing the requirement for the endogenous VGD1 PME for growth of Arabidopsis pollen tubes (Jiang et al., 2005) and another reporting inhibition of growth of pollen tubes of Lilium formosanum by exogenously added tobacco PME, NtPPME1 (Bosch et al., 2005). One possible complication in interpretation of the PME function in pollen is that both PMEs with a demonstrated role in the pollen tube growth, VGD1 and NtPPME1, contain, in addition to their catalytic PME domain, a PMEI domain. This domain, which is similar to the kiwi (Camardella et al., 2000; Giovane et al., 1995) and Arabidopsis PME inhibitors (Raiola et al., 2004; Wolf et al., 2003), PMEIs, most likely impairs the enzymatic activity of the PME domain by intramolecular

Fig. 5. Effects of AtPPME1 on morphology of in vitro germinated pollen tubes. (A) A wild-type pollen tube 6 h post-germination. (BD) Unusually shaped and stunted atppme1 pollen tubes 6 h post-germination. Scale bars = 10 m. (E) Wild-type pollen tubes 24 h post germination. (F) Stunted atppme1 pollen tubes 24 h post germination. (G) Pollen tubes from atppme1 plants genetically complemented with the wild-type AtPPME1 gene 24 h post-germination. Scale bars = 50 m. (H) A wild-type pollen tube tip 24 h post-germination. (I) Unusually shaped atppme1 pollen tube tip 24 h post-germination. (J) A pollen tube tip from atppme1 plants genetically complemented with the wild-type AtPPME1 gene 24 h post-germination. Scale bars = 5 m.

from the wild-type pollen tubes (compare Figs. 5G to E and Figs. 5J to H). The average growth rate of these transgenic pollen tubes (33.4 5.7 m/h, n = 150) was also similar to that of the pollen tubes from the wild-type plants. Furthermore, the total PME activity in the pollen grains of atppme1/AtPPME1 plants was restored to the wild-type levels (Fig. 4C). Thus, the atppme1 phenotype is most likely due to disruption of the native AtPPME1 function by the mutation in this gene.

Fig. 6. Similar morphology of wild-type and atppme1 pollen grains. (A) Wildtype pollen grains. (B) Pollen grains from atppme1 plants. Insets show higher magnification of the pollen grain cell wall. Cell walls were stained with propidium iodide, the signal of which is in red. All images are projections of several confocal sections. Scale bars = 5 m.

90

G.-W. Tian et al. / Developmental Biology 294 (2006) 8391

inhibition (Bosch et al., 2005). Thus, the apparent activity of PMEI-containing PMEs and their biological effects most likely represents a result of complex interplay of both the enzyme and its inhibitor. To circumvent this difficulty and to obtain a direct insight into the role of PME in pollen tube growth, it would be useful to study a PME gene that encodes only the PME domain, but not the PMEI domain. Our analysis of the Arabidopsis genome sequence identified 23 such PMEs among the 66 members of the PME-like protein family. We selected one of these genes, designated it AtPPME1, and demonstrated that its expression is pollen-specific. The specificity of AtPPME1 expression was determined using the FTFLP approach which offers two significant advantages for studies of gene expression: first, it yields information about the tissue specificity of gene expression by the use of native promoters, and second, it produces internally tagged full-length proteins that are likely to exhibit native intracellular localization (Tian et al., 2004). Indeed, our results demonstrated tight restriction of the AtPPME1 gene expression to pollen grains and pollen tubes and showed that the AtPPME1 protein accumulates in the cell periphery and intracellular structures most likely representing the endomembrane system known to be involved in secretion of PMEs (e.g., Bosch et al., 2005; Gaffe et al., 1997). Thus, AtPPME1 represented a new pollen-specific PME which has not been discovered by the previous study of the Arabidopsis pollen transcriptome (Pina et al., 2005). The biological function of AtPPME1 was then studied by reverse genetics using a T-DNA insertional mutant of the atppme1 gene. The atppme1 mutation is a novel mutation which compromises the shape and growth rate of Arabidopsis pollen tubes, but, unlike the vgd1 mutation (Jiang et al., 2005), does not affect the pollen tube stability, silique morphology, and plant fertility. Thus, AtPPME1 may have a more specific, narrowly defined function in pollen tube growth than VGD1, indicating functional differences between different pollen-specific PMEs. The slow growth and irregular shape of the atppme1 pollen tubes are most likely due to a reduced rigidity of their cell walls. This, in turn, may result from the reduction of the loss of the AtPPME1 activity in the mutant. Generally, PMEs demethylesterify pectin chains either randomly or processively. The mode of the PME action is thought to depend on the isoelectric point (pI) of the enzyme, with acidic PMEs acting randomly and basic PMEs functioning processively or linearly (Micheli, 2001). Because AtPPME1 is a basic molecule with a predicted pI of 8.7 (Table 1), it is most likely a processive enzyme, demethylesterifying stretches of homogalacturonan molecules in the pollen tube walls and creating regions of free carboxyl groups. These can interact with calcium cations, altering their local concentration around the growing pollen tubes. Because normal pollen tube growth is associated with a specific concentration of Ca2+ (Franklin-Tong, 1999; Malho et al., 2000; Messerli et al., 2000; Trewavas and Malho, 1998), alterations of this concentration due to the lack of the AtPPME1 enzymatic activity in the atppme1 mutant may compromise the structure and elongation rates of the mutant pollen tubes. Thus, AtPPME1 likely plays an important and specific role in normal

growth and morphological development of pollen tubes in Arabidopsis. The biological significance of this role is underscored by the observations that a relatively small effect of the lack of AtPPME1 on the total PME activity of the pollen causes profound functional consequences, bringing about dramatic changes in the pollen tube growth and development. Acknowledgments Our laboratory is supported by grants from the National Institutes of Health, the National Science Foundation, the National Research Initiative of the U.S. Department of Agriculture, the U.S.Israel Binational Agricultural Research and Development Fund (BARD), and the U.S.Israel Binational Science Foundation to V.C., and by Vaadia-BARD Postdoctoral Fellowship Award to A.Z. References
Albani, D., Altosaar, I., Arnison, P.G., Fabijanski, S.F., 1991. A gene showing sequence similarity to pectin esterase is specifically expressed in developing pollen of Brassica napus. Sequences in its 5 flanking region are conserved in other pollen-specific promoters. Plant. Mol. Biol. 16, 501513. Alonso, J.M., Stepanova, A.N., Leisse, T.J., Kim, C.J., Chen, H., Shinn, P., Stevenson, D.K., Zimmerman, J., Barajas, P., Cheuk, R., Gadrinab, C., Heller, C., Jeske, A., Koesema, E., Meyers, C.C., Parker, H., Prednis, L., Ansari, Y., Choy, N., Deen, H., Geralt, M., Hazari, N., Hom, E., Karnes, M., Mulholland, C., Ndubaku, R., Schmidt, I., Guzman, P., Aguilar-Henonin, L., Schmid, M., Weigel, D., Carter, D.E., Marchand, T., Risseeuw, E., Brogden, D., Zeko, A., Crosby, W.L., Berry, C.C., Ecker, J.R., 2003. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653657. An, Y.Q., McDowell, J.M., Huang, S., McKinney, E.C., Chambliss, S., Meagher, R.B., 1996. Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues. Plant J. 10, 107121. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Smith, J.A., Seidman, J. G., Struhl, K., 1987. Current Protocols in Molecular Biology. Greene Publishing-Wiley Interscience, New York, NY. Bordenave, M., Breton, C., Goldberg, R., Huet, J.C., Perez, S., Pernollet, J.C., 1996. Pectinmethylesterase isoforms from Vigna radiata hypocotyl cell walls: kinetic properties and molecular cloning of a cDNA encoding the most alkaline isoform. Plant Mol. Biol. 31, 10391049. Bosch, M., Cheung, A.Y., Hepler, P.K., 2005. Pectin methylesterase, a regulator of pollen tube growth. Plant Physiol. 138, 13341346. Bourgault, R., Bewley, J.D., 2002. Gel diffusion assays for endo-betamannanase and pectin methylesterase can underestimate enzyme activity due to proteolytic degradation: a remedy. Anal. Biochem. 300, 8793. Brummell, D.A., Harpster, M.H., 2001. Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. Plant Mol. Biol. 47, 311340. Camardella, L., Carratore, V., Ciardiello, M.A., Servillo, L., Balestrieri, C., Giovane, A., 2000. Kiwi protein inhibitor of pectin methylesterase aminoacid sequence and structural importance of two disulfide bridges. Eur. J. Biochem. 267, 45614565. Catoire, L., Pierron, M., Morvan, C., du Penhoat, C.H., Goldberg, R., 1998. Investigation of the action patterns of pectinmethylesterase isoforms through kinetic analyses and NMR spectroscopy. Implications In cell wall expansion. J. Biol. Chem. 273, 3315033156. Chen, M.H., Citovsky, V., 2003. Systemic movement of a tobamovirus requires host cell pectin methylesterase. Plant J. 35, 386392. Chen, M.H., Tian, G.W., Gafni, Y., Citovsky, V., 2005. Effects of calreticulin on viral cell-to-cell movement. Plant Physiol. 138, 18661876. Dens, J.M., Baron, A., Renard, C.M., Pean, C., Drilleau, J.F., 2000. Different action patterns for apple pectin methylesterase at pH 7.0 and 4.5. Carbohydr. Res. 327, 385393.

G.-W. Tian et al. / Developmental Biology 294 (2006) 8391 Downie, B., Dirk, L.M., Hadfield, K.A., Wilkins, T.A., Bennett, A.B., Bradford, K.J., 1998. A gel diffusion assay for quantification of pectin methylesterase activity. Anal. Biochem. 264, 149157. Ferguson, C., Teeri, T.T., Siika, A.M., Read, S.M., Bacic, A., 1998. Location of cellulose and callose in pollen tubes and grains of Nicotiana tabacum. Planta 206, 452460. Franklin-Tong, V.E., 1999. Signaling in pollination. Curr. Opin. Plant Biol. 2, 490495. Frenkel, C., Peters, J.S., Tieman, D.M., Tiznado, M.E., Handa, A.K., 1998. Pectin methylesterase regulates methanol and ethanol accumulation in ripening tomato (Lycopersicon esculentum) fruit. J. Biol. Chem. 273, 42934295. Futamura, N., Mori, H., Kouchi, H., Shinohara, K., 2000. Male flower-specific expression of genes for polygalacturonase, pectin methylesterase and beta1,3-glucanase in a dioecious willow (Salix gilgiana Seemen). Plant Cell Physiol. 41, 1626. Gaffe, J., Tiznado, M.E., Handa, A.K., 1997. Characterization and functional expression of a ubiquitously expressed tomato pectin methylesterase. Plant Physiol. 114, 15471556. Giovane, A., Balestrieri, C., Quagliuolo, L., Castaldo, D., Servillo, L., 1995. A glycoprotein inhibitor of pectin methylesterase in kiwi fruit. Purification by affinity chromatography and evidence of a ripening-related precursor. Eur. J. Biochem. 233, 926929. Griesbeck, O., Baird, G.S., Campbell, R.E., Zacharias, D.A., Tsien, R.Y., 2001. Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J. Biol. Chem. 276, 2918829194. Jiang, L., Yang, S.L., Xie, L.F., Puah, C.S., Zhang, X.Q., Yang, W.C., Sundaresan, V., Ye, D., 2005. VANGUARD1 encodes a pectin methylesterase that enhances pollen tube growth in the Arabidopsis style and transmitting tract. Plant Cell 17, 584596. Johnson-Brousseau, S.A., McCormick, S., 2004. A compendium of methods useful for characterizing Arabidopsis pollen mutants and gametophyticallyexpressed genes. Plant J. 39, 761775. Kang, J., Kuhn, J.E., Schafer, P., Immelmann, A., Henco, K., 1995. Quantification of DNA and RNA by PCR. In: McPherson, M.J., Hames, B.D., Taylor, G.R. (Eds.), PCR 2, A Practical Approach. IRL Press, Oxford, pp. 119133. Kim, J.Y., Yuan, Z., Jackson, D., 2003. Developmental regulation and significance of KNOX protein trafficking in Arabidopsis. Development 130, 43514362. Lacoux, J., Duval, I., Dupre, P., Gutierrez, L., Lesueur, S., Roger, D., Laine, E., 2003. Activity of a flax pectin methylesterase promoter in transgenic tobacco pollen. J. Plant Physiol. 160, 977979. Letunic, I., Copley, R.R., Schmidt, S., Ciccarelli, F.D., Doerks, T., Schultz, J., Ponting, C.P., Bork, P., 2004. SMART 4.0: towards genomic data integration. Nucleic Acids Res. 32, D142D144. Li, Y.Q., Chen, F., Linskens, H.F., Cresti, M., 1994. Distribution of unesterified and esterified pectins in cell wall of pollen tubes. Sex. Plant Reprod. 7, 145152. Li, J., Krichevsky, A., Vaidya, M., Tzfira, T., Citovsky, V., 2005. Uncoupling of the functions of the Arabidopsis VIP1 protein in transient and stable plant genetic transformation by Agrobacterium. Proc. Natl. Acad. Sci. U. S. A. 102, 57335738. Lord, E.M., Russell, S.D., 2002. The mechanisms of pollination and fertilization in plants. Annu. Rev. Cell Dev. Biol. 18, 81105. Malho, R., Camacho, L., Moutinho, A., 2000. Signalling pathways in pollen tube growth and reorientation. Ann. Bot. 85, 5968. Markovic, O., Jornvall, H., 1992. Disulfide bridges in tomato pectinesterase: variations from pectinesterases of other species; conservation of possible active site segments. Protein Sci. 1, 12881292. Messerli, M.A., Creton, R., Jaffe, L.F., Robinson, K.R., 2000. Periodic increases in elongation rate precede increases in cytosolic Ca2+ during pollen tube growth. Dev. Biol. 222, 8498. Micheli, F., 2001. Pectin methylesterases: cell wall enzymes with important roles in plant physiology. Trends Plant Sci. 6, 414419. Ni, M., Tepperman, J.M., Quail, P.H., 1998. PIF3, a phytochrome-interacting

91

factor necessary for normal photoinduced signal transduction, is a novel basic helixloophelix protein. Cell 95, 657667. Palanivelu, R., Preuss, D., 2000. Pollen tube targeting and axon guidance: parallels in tip growth mechanisms. Trends Cell Biol. 10, 517524. Pilling, J., Willmitzer, L., Fisahn, J., 2000. Expression of a Petunia inflata pectin methyl esterase in Solanum tuberosum L. enhances stem elongation and modifies cation distribution. Planta 210, 391399. Pina, C., Pinto, F., Feijo, J.A., Becker, J.D., 2005. Gene family analysis of the Arabidopsis pollen transcriptome reveals biological implications for cell growth, division control, and gene expression regulation. Plant Physiol. 138, 744756. Plastow, G.S., 1988. Molecular cloning and nucleotide sequence of the pectin methyl esterase gene of Erwinia chrysanthemi B374. Mol. Microbiol. 2, 247254. Preuss, D., 2002. Sexual signaling on a cellular level: lessons from plant reproduction. Mol. Biol. Cell 13, 18031805. Qiu, X., Erickson, L., 1995. A pollen-specific cDNA (P65, Accession No. U28148) encoding a putative pectin esterase in alfalfa (PGR95-094). Plant Physiol. 109, 1127. Raiola, A., Camardella, L., Giovane, A., Mattei, B., De Lorenzo, G., Cervone, F., Bellincampi, D., 2004. Two Arabidopsis thaliana genes encode functional pectin methylesterase inhibitors. FEBS Lett. 557, 199203. Ray, J., Knapp, J., Grierson, D., Bird, C., Schuch, W., 1988. Identification and sequence determination of a cDNA clone for tomato pectin esterase. Eur. J. Biochem. 174, 119124. Rodrguez-Llorente, I.D., Prez-Hormaeche, J., Mounadi, K.E., Dary, M., Caviedes, M.A., Cosson, V., Kondorosi, A., Ratet, P., Palomares, A.J., 2004. From pollen tubes to infection threads: recruitment of Medicago floral pectic genes for symbiosis. Plant J. 39, 587598. Rogers, H.J., Bate, N., Combe, J., Sullivan, J., Sweetman, J., Swan, C., Lonsdale, D.M., Twell, D., 2001. Functional analysis of cis-regulatory elements within the promoter of the tobacco late pollen gene g10. Plant Mol. Biol. 45, 577585. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406425. Schultz, J., Milpetz, F., Bork, P., Ponting, C.P., 1998. SMART, a simple modular architecture research tool: identification of signaling domains. Proc. Natl. Acad. Sci. U. S. A. 95, 58575864. Taylor, L.P., Hepler, P.K., 1997. Pollen germination and tube growth. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 461491. The Arabidopsis Genome Initiative, 2000. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796815. Thorsness, M.K., Kandasamy, M.K., Nasrallah, M.E., Nasrallah, J.B., 1993. Genetic ablation of floral cells in Arabidopsis. Plant Cell 5, 253261. Tian, G.W., Mohanty, A., Chary, S.N., Li, S., Paap, B., Drakakaki, G., Kopec, C.D., Li, J., Ehrhardt, D., Jackson, D., Rhee, S., Raikhel, N., Citovsky, V., 2004. High-throughput fluorescent tagging of full-length Arabidopsis gene products in planta. Plant Physiol. 135, 2538. Trewavas, A.J., Malho, R., 1998. Ca2+ signalling in plant cells: the big network! Curr. Opin. Plant Biol. 1, 428433. Tzfira, T., Tian, G.W., Lacroix, B., Vyas, S., Li, J., Leitner-Dagan, Y., Krichevsky, A., Taylor, T., Vainstein, A., Citovsky, V., 2005. pSAT vectors: a modular series of plasmids for fluorescent protein tagging and expression of multiple genes in plants. Plant Mol. Biol. 57, 503516. Wakeley, P.R., Rogers, H.J., Rozycka, M., Greenland, A.J., Hussey, P.J., 1998. A maize pectin methylesterase-like gene, ZmC5, specifically expressed in pollen. Plant Mol. Biol. 37, 187192. Walhout, A.J., Temple, G.F., Brasch, M.A., Hartley, J.L., Lorson, M.A., van den Heuvel, S., Vidal, M., 2000. GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol. 328, 575592. Wen, F., Zhu, Y., Hawes, M.C., 1999. Effect of pectin methylesterase gene expression on pea root development. Plant Cell 11, 11291140. Wolf, S., Grsic-Rausch, S., Rausch, T., Greiner, S., 2003. Identification of pollen-expressed pectin methylesterase inhibitors in Arabidopsis. FEBS Lett. 555, 551555.